In a recent study posted to the bioRxiv* preprint server, researchers discovered and optimized cysteine-reactive pyrazoline-based covalent inhibitors for several coronaviruses' main protease (Mpro), including severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).
The constantly mutating spike (S) protein alters and reduces the effectiveness of currently used vaccines and antibody therapeutics targeting SARS-CoV-2. Among several genes and proteins conserved across coronaviruses, the Mpro or 3C-like protease (3CLpro) is undeniably the most attractive target for developing antiviral therapies.
The Food and Drug Association (FDA)-approved oral drug combination of ritonavir-boosted nirmatrelvir, marketed as Paxlovid, is a Mpro inhibitor and is widely used for coronavirus disease 2019 (COVID-19) treatment. Despite the availability of Paxlovid, there is still room for discovering more Mpro inhibitors that may be used for drug discovery and development efforts, leading to even more efficacious treatments. Additionally, there may be the emergence of resistance against Paxlovid, requiring additional Mpro inhibitors.
The Mpro enzyme acts as a catalyst for several proteolytic processing events facilitating replication of coronaviruses inside the host cells. Since cysteine (C145) drives the catalytic activity of Mpro, cysteine-reactive pyrazoline-based covalent ligand naturally appear attractive for developing Mpro inhibitors that can irreversibly interact with the Mpro C145. Overall, inhibiting Mpro with an antiviral could effectively prevent coronaviruses from replicating, providing therapeutic benefit.
About the study
In the current study, researchers used gel-based activity-based protein profiling (ABPP)-based approaches to screen a library of 582 acrylamides and chloroacetamides. The cysteine-reactive rhodamine-functionalized iodoacetamide probe (IA-rhodamine) competed against cysteine-reactive covalent ligands in the ABPP screen.
Next, they tested four pyrazoline-based chloroacetamide ligands EN71, EN82, EN216, and EN223 in a fluorescence resonance energy transfer (FRET)-based activity assay employing a Mpro peptide substrate to identify compounds that inhibited Mpro activity. To detect potent inhibitors with 50% inhibitory concentration (IC50) values <100nM, they switched over from the FRET-based peptide probe to a rhodamine-based Mpro substrate activity assay which required Mpro concentrations of less than 115 nM.
The researchers also performed chemoproteomic profiling of EN82 cysteine-reactivity in HEK293T cell lysate to assess whether this compound demonstrated some degree of selectivity or whether it was non-specific. Furthermore, using a rapid mass spectrometry-based covalent chemoproteomics workflow, they quantitatively analyzed EN82-competed cysteine sites. HEK293T cell lysates spiked with SARS-CoV-2 Mpro were pre-treated with vehicle or 10 µM of EN82 and subsequently labeled with acid-cleavable cysteine-reactive iodoacetamide-based enrichment probe.
The researchers also performed structure-activity relationship (SAR) studies around the central pyrazoline core to optimize the potency of the discovered chemical scaffolds.
Lastly, they used an Agilent RapidFire-based substrate peptide activity assay (more sensitive and required much-less Mpro protein compared to the rhodamine-based activity assays) to comparatively assess the potencies of the best covalent ligands discovered during the study. Likewise, they tested several di- and tri-substituted compounds, including PM-2-071, against a panel of Mpro enzymes from SARS-CoV-2 and other former coronaviruses.
Pyrazoline-based chloroacetamide ligands EN71, EN82, EN216, and EN223 demonstrated dose-dependent inhibition of Mpro IA-rhodamine labeling. Of all the four ligands, the pyrazoline EN82 displayed the highest potency against the SARS-CoV-2 Mpro with an apparent IC50 value of 0.16 µM (for an incubation time of 30 minutes) compared to 0.52-4.8 µM for EN216, EN71, and EN223.
On rhodamine-based assay, EN82 showed apparent IC50 values of 0.091 µM, 0.059 µM, and 0.56 µM against Mpro from SARS-CoV-2, SARS-CoV-1, and Middle Eastern respiratory syndrome coronavirus (MERS-CoV), respectively.
Mpro C145 emerged as the primary target of EN82 with only one off-target HMOX2 C282 from more than 1000 distinct quantified probe-modified cysteines, thus suggesting that it had a high degree of proteome-wide selectivity.
Most of the compounds tested by Agilent RapidFire-based substrate peptide activity assay inhibited Mpro from several coronaviruses. The apparent IC50 values in these assays were in the nanomolar range; for instance, PM-2-071 had IC50s <2nM across SARS-CoV-2, human coronavirus HKU1 (HCoV-HKU1), and human coronavirus OC43 (HCoV-OC43). Thus, the PM-2-071 scaffold emerged as a promising candidate for further optimization as a pan-coronavirus Mpro inhibitor.
SAR explorations of the pyrazoline core revealed the relative stereochemistry at the C4 and C5 carbons. Although some of the optimized inhibitors, such as PM-2-071 had issues related to metabolic stability, solubility, and cell permeability and exhibited no antiviral activity against SARS-CoV-2; however, 5-chloro-2-fluorophenyl C3-substituent (CMZ-53), a 3,5-disubstituted pyrazoline provided a nearly three-fold improvement in potency compared to EN82.
The present study discovered highly potent SARS-CoV-2 Mpro inhibitors based on pyrazoline-based chloroacetamides and vinyl sulfonamides. In the future, further optimization of these potent Mpro inhibitors could enhance their antiviral efficacy and promote their drug-like properties. Nonetheless, these cysteine-reactive warheads represented a solid starting point for developing more advanced non-peptidic and more drug-like pan-coronavirus Mpro inhibitors with remarkable potencies.
bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.